Lysing reagent, cartridge and automatic electronic cell counter for simultaneous enumeration of different types of white blood cells

ABSTRACT

The present invention relates to a lysing reagent for use in the simultaneous automatic electronic enumeration and volumetric discrimination of different types of blood cells, such as leukocytes, thrombocytes, etc, in blood. Further, the present invention relates to a Coulter (impedance) counting apparatus containing the lysing reagent, for example a Coulter counting apparatus with a disposable cartridge for characterizing cells suspended in a liquid, especially a self-contained disposable cartridge for single-use analysis, such as for single-use analysis of a small quantity of whole blood.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. 119 to Danishapplication PA 2003 00159 filed on Feb. 5, 2003, and under 35 U.S.C.119(e)(1) to U.S. Provisional application Ser. No. 60/387,407 filed onJun. 11, 2002, which are hereby incorporated by reference.

A lysing reagent for the simultaneous enumeration of different types ofwhite blood cells.

The present invention relates to a lysing reagent for use in thesimultaneous automatic electronic enumeration and volumetricdiscrimination of different types of blood cells, such as leukocytes,thrombocytes, etc, in blood. Further, the present invention relates to aCoulter (impedance) counting apparatus containing the lysing reagent,for example a Coulter counting apparatus with a disposable cartridge forcharacterizing cells suspended in a liquid, especially a self-containeddisposable cartridge for single-use analysis, such as for single-useanalysis of a small quantity of whole blood.

The three types of blood cells erythrocytes, leukocytes and thrombocytesdiffer in a) size, b) occurrence and c) structure.

a) Their size, expressed as diameter, ranges from 2 μm for the smallestthrombocytes to 20 μm for the largest leukocytes.

b) Their occurrence normally ranges from typically 4.109/L blood forleukocytes to typically 4.1012/L blood for erythrocytes.

c 1) The thrombocytes contain organelles such as microfilaments,granules (including so called dense-bodies) and mitochondria, surroundedby a cell membrane. This membrane is in its turn surrounded by anso-called surface coat.

c 2) The most distinct leukocyte organelle is the microscopicallyvisible granule, which has different structure in the differentleukocyte sub-populations.

c 3) The erythrocytes cell membrane does, however, not surround anyorganelles as mentioned above. Finally, the three types of blood cellshave different shape. (Clinical Haematology, Ed. M. M. Wintrobe, 1981,8th Ed, Lia & Febiger, Philadelphia, USA)

Information on the content of leukocytes, their subpopulations andthrombocytes is an important tool for the physician in order to diagnosedifferent diseases and monitor treatment. Furthermore, the concentrationof haemoglobin, directly related to the number of erythrocytes, in theblood sample is also of great importance.

Thus, much effort has over the years been devoted to the development ofautomated blood cell counting systems. For the sake of completeness,manual counting using microscope techniques of blood cells using smearsof blood samples should also be mentioned here. The latter techniquesare, however, time-consuming and require relatively high level oftraining by the performer. Automated blood cell counting systems can bedivided into two major groups: those relying on the impedance cellsizing principle (equal to the Coulter principle) and those relying onthe flow cytometry principle. Examples of such automated systems areCoulter AcT Diff based on impedance cell sizing and Bayer ADVIA 120based on flow cytometry. Both principles can be combined withspectrophotometric techniques for additional analysis of solublecomponents in the blood, such as haemoglobin.

The automated blood cell counting systems analyse the number oferythrocytes and thrombocytes simultaneously using a special procedureinvolving dilution of the blood sample with cell-preserving reagent. Thenumber of leukocytes are on the other hand separately analysed byexposing the blood sample to a lysing reagent containing compoundscapable of lysing the erythrocytes since they interfere with theenumeration of the leukocytes. The interference, not usingerythrocyte-lysing compounds, is due to the overlap in cell size and itis thus not possible to count the leukocytes in the presence of theerythrocytes by size discrimination alone.

The lysing reagent does also affect the leukocytes but only to such anextent that these can still be enumerated. As a consequence of lysingthe erythrocytes, the haemoglobin is released and can thus be properlyanalysed using spectrophotometric techniques. A great number of(erythrocyte) lysing reagents mixtures have been developed. In thesemixtures specific compounds, surfactants, are responsible for the lysingaction. Examples of such compounds are quaternary ammonium salts asdescribed in U.S. Pat. No. 4,485,175, U.S. Pat. No. 4,346,018, U.S. Pat.No. 4,745,071, U.S. Pat. No. 4,528,274, U.S. Pat. No. 5,763,280, U.S.Pat. No. 5,834,315 and saponins as described in U.S. Pat. No. 4,751,179,U.S. Pat. No. 5,731,206, U.S. Pat. No. 5,840,515, U.S. Pat. No.5,348,859, EP 0549 414 B1. Different lysing reagent in the automaticblood cell counting systems as described above have different effect onthe system to enumerate the three leukocyte subpopulations: lymphocytes,monocytes and granulocytes. In order to also be able to enumerate thethree granulocytes sub-populations: eososinophils, basofis andneutrophils the lysing reagent also contains specific compoundsrendering these three subpopulations different properties. As analternative these three granulocytes subpopulations can be enumeratedusing complementary radio frequency analysis contained in the automatedblood cell counting systems based on the impedance cell sizingprinciple.

In EP 0549 414 B1, a process is disclosed wherein a combined enumerationof erythrocytes and thrombocytes in a blood sample is performed followedby addition of an erythrocyte lysing agent whereafter the remainingthrombocytes are enumerated again. Finally, the number of theerythrocytes and the thrombocytes, respectively, is calculated.

According to the present invention, an improved method and apparatus isprovided for enumeration of different types of blood cells, such asdifferent types of white blood cells, such as leukocytes, thrombocytes,etc, in blood. The method and apparatus involve counting anddistinguishing the different types in one counting operation, e.g. in aflowcytometer, in a Coulter counter, etc. A lysing reagent is added to ablood sample for lysing of the erythrocytes. The composition of thelysing reagent is accurately controlled so that the required lysing isperformed without deteriorating the cell types that it is desired toenumerate. For example, in a Coulter counter, the diameter of theorifice is optimised for counting of cell sizes corresponding to thecell sizes of the different types of cells it is desired to enumerateafter lysing by the lysing reagent.

For example, the lysing reagent according to the present invention maybe contained in a cartridge utilised in an apparatus for enumeration ofcells in a blood sample, comprising a housing with a first mixingchamber and a first collection chamber separated by a wall containing anorifice for passage of the cells between the first mixing chamber andthe first collection chamber. Cell characterization means are providedfor characterizing cells passing through the orifice. The apparatuscomprises a docking station for removably receiving the cartridge, thedocking station comprising connectors for operational connection withthe cell characterization means when the cartridge is received in thedocking station.

With the electrical impedance technique of the Coulter counterprinciple, it is possible to resolve the cell volume from themeasurement. By maintaining a constant current across the orifice, therecorded voltage pulse from cells displacing the electrolyte in theorifice will have a height proportional to the volume of the cell. Thisis because cells can be considered non-conducting compared to theelectrolyte, the electrical field (DC or RF) in the centre of theorifice is homogeneous, which is normally the case when the diameter Dis smaller than the length l of the orifice (l/D>1), the cell d is to beconsidered small compared to the diameter of the orifice (d<0.2*D), onlyone cell passes through at a time and the cells are passed through theorifice along the length or depth of the orifice.

Preferably, the length of the orifice is from 1 to 1000 μm, for exampleabout 50 μm. Desirably the length of the orifice is chosen such thatonly one cell will be present in the orifice at the time when detectingcells of from 0.1 to 100 μm diameter. However, considerations to thehomogeneity of the electrical field in the orifice may require a lengthof the orifice larger or equal to the diameter. The counts, of whichsome may be simultaneous counting of two cells, can be correctedmathematically by implementing a statistical estimation. The aspectratio of the orifice, (length or depth divided by diameter) ispreferably from 0.5:1 to 5:1, more preferably from 1:1 to 3:1.

Preferably, the largest cross-sectional dimension of the orifice is from5 to 200 μm, for example 10 to 50 μm.

As a supplement a spectrophotometric measurement can be performed inorder to quantify the content of e.g. haemoglobin.

The lysing reagent according to the present invention affects varioustypes of blood cells. For example, the erythrocytes are completelyeliminated, the leukocytes decrease in volume and the thrombocytes alsodecrease in volume. However, the composition of the lysing reagent isaccurately controlled so that the decrease in volume of the thrombocytesis not significant for the ability of the thrombocytes to be counted ina Coulter counter. It has been verified that the identified parts of theremains of the thrombocytes were related to the original content ofthrombocytes in the blood samples according to comparative methods. Thisis further described below.

The different behaviour of the different blood cells when exposed to thelysing reagent in question, can be explained taken the differentstructures of the three types of blood cells, as outlined above, intoaccount and its consequence on the remains of these after exposure tothe lysing reagent. The remains of the leukocytes are made up of thegranula possibly surrounded by a collapsed cell membrane and/or adecreased amount of cytoplasm. The erythrocyte cell membranedisintegrates completely during the action of the lysing reagent. Sincethe erythrocytes do not contain any cell organelles, small cell membranedebris is the only remains of this blood cell type. This debris isdifficult to separate from the background-noise of automatic blood cellcounting systems based on the impedance cell sizing. The remains of thethrombocytes after exposure to the lysing reagent could be made up ofcell organelles possibly surrounded by a collapsed cell membrane aftercomplete disintegration of the outer surface coat.

In order to further separate the remains of the thrombocytes from thebackground-noise we found that use of an automatic blood cell system,based on impedance cell sizing principle, with a smaller orifice can bemade. Using an orifice with a diameter of 50 μm, 5% of the thrombocytespresent in the sample can thus be accounted for. This part of thethrombocytes can be increased up to 49% using an orifice with a diameterof 44 μm. Finally, using an orifice with a diameter of 40 μm, 63% of thein the sample present thrombocytes can be accounted for.

The cartridge may comprise the following parts:

1. A liquid storage chamber

2. A blood-sampling device

3. A first mixing chamber

4. A flow through sensor arrangement

5. A first collection chamber

6. A volume metering arrangement comprised of a chamber and twoconnected flow channels

7. A hydraulic connection for moving the liquid through the cartridge

The concept of the disposable unit can be further combined with thefollowing additional parts:

A. Optical structures for optical liquid level measurement

B. Electrodes for liquid level measurement

C. Anti-coagulation treatment of surfaces

D. Reagents in the diluent for modification of e.g. blood cells

E. Mixing flee or baffle for assisted mixing

F. Multiple volume metering arrangements for altering volumes

G. A coating tape covering the sample inlet before use

H. A waste chamber for waste/overflow

I. A valve preventing liquid to exit through exhaust tube

J. A piston or membrane instead of the vacuum tube

K. A window for spectrophotometric measurements

The liquid storage chamber (part 1) holds the required amount of diluentused for the blood analysis. When the blood has been sampled into thecartridge, the diluent is flushed through the capillary to wash out thesampled blood and dilute it as required by the test. Dilutions of 100 to100,000 times are considered to be normal ratings and dilutions of 500to 10,000 times are preferred. The liquid storage chamber shouldpreferably be constructed to facilitate total draining of the chamber.This would be accomplished by having a slanting of the bottom of thechamber.

The sampling unit (part 2) may comprise a capillary extending through amovable rod placed in a tight-fitting supporting body. The movable rodis used for entrapment of a precise amount of blood sample. When bloodhas filled the capillary by capillary forces, the rod is turned and/ordisplaced from its initial position in the supporting body, thusisolating the part of the capillary that extends through the rod.

After moving the rod in the supporting body into its second position thecapillary forms a liquid path between the liquid storage chamber and thefirst mixing chamber (part 3). By applying a low pressure to the firstmixing chamber the diluent and blood sample is forced into the firstmixing chamber, where mixing will be performed by convection orsubsequently by blowing bubbles into the mixing chamber.

The flow through sensor arrangement (part 4) is comprised of a smallorifice in a membrane that establishes a liquid path from the firstmixing chamber to the first collection chamber. On each side of themembrane (in the first mixing chamber and in the first collectionchamber) an electrode is placed contacting the liquid.

The first collection chamber (part 5) forms a liquid priming function ofthe backside of the sensor system.

The volume metering system (part 6) is necessary for determination ofthe cell concentration. It comprises volume-metering chamber of a knownvolume with two relatively thin channels connecting the inlet at thebottom and the outlet at the top. Sensing of the liquid at the inlet andoutlet can be applied by optical or electrical means.

The outlet of the volume metering system is connected through a channel(part 7) to a source of pressure for moving the liquid through thecartridge.

The additional parts to the concept are further described here:

Addition A: Optical detection by change of optical properties of achannel such as changed reflectance or transmittance due to replacementof air with liquid in the channel. The surface over the inlet and outletof the volume-metering cell should be structured to optimize thecoupling of the light into the channel. The presence of liquid in atransparent polymer channel will result in a transmission of the signalsas opposed to a reflection when no liquid is present, which can beregistered by optical sensors.

Addition B: Two electrodes for liquid level measurement are connectedthrough the body of the cartridge into the inlet and outlet of thevolume-metering cell respectively. The electrodes will beshort-circuited through the saline liquid to the electrode placed in thefirst collection chamber, which can be registered through an externalelectrical arrangement.

Addition C: The anti-coagulation treatment of surfaces in the samplingstructure can be achieved by having selected compounds adhered orchemically bonded to these surfaces. Examples of such compounds areheparin and salts of EDTA.

Addition D: Reagent in the diluent for modification of e.g. blood cells.This reagent can consist of one or several compounds capable ofhemolysing the erythrocytes. In addition other compounds may be added inorder to: stabilize leukocytes and/or thrombocytes, adjust the pH-valueand osmotic pressure, minimize bacterial growth, modify the haemoglobinpresent and minimize batch to batch variations. The following exampleshave been included to provide information on relevant subjects relatedto the performance of a self-contained test cartridge.

Examples of compounds capable of selectively hemolysing the red bloodcells are: mixtures of quaternary ammonium salts as described in e.g.U.S. Pat. No. 4,485,175; U.S. Pat. No. 4,346,018; U.S. Pat. No.4,745,071; U.S. Pat. No. 4,528,274; and U.S. Pat. No. 5,834,315.

Examples of compounds capable of, during the hemolysis of the red bloodcells, stabilizing the leukocytes are N-(1-acetamido)iminodiacetic acid,procaine hydrochloride as described in e.g. U.S. Pat. No. 4,485,175 and1,3-dimethylurea as described in e.g. U.S. Pat. No. 4,745,071. Inaddition N-(1-acetamido)iminodiacetic acid is proposed to further assistthe quaternary ammonium salts in minimizing debris stemming fromhemolysed red blood cells as described in e.g. U.S. Pat. No. 4,962,038and adjust the pH-value (see below).

Examples of compounds added in order to adjust the pH-value and notleast importantly the osmotic pressure of the diluent are:N-(1-acetamido)iminodiacetic acid, sodium chloride, sodium sulphate asdescribed in e.g. U.S. Pat. No. 4,485,175 and U.S. Pat. No. 4,962,038.

Examples of compounds capable of minimizing bacterial growth are:1,3-dimethylolurea and chlorhexidine diacetate as described in e.g. U.S.Pat. No. 4,962,038.

Examples of compounds added to convert the haemoglobin species to anend-product suitable for spectrophotometric analysis are: potassiumcyanide as described in e.g. U.S. Pat. No. 4,485,175; U.S. Pat. No.4,745,071; U.S. Pat. No. 4,528,274 and tetrazole or triazole asdescribed in WO 99/49319.

Examples of cells or compounds which may be added in order to introducea tool for minimizing variation between different batches of thedisposable device are: latex beads of known size and glass beads ofknown size.

Addition E: If assisted mixing is required the first mixing chambermight optionally include a mixing flee or a baffle. A magnetic flee maybe used to force the convection through an externally moving magneticfield. A baffle may be used to mechanically stir the liquid when movedby an externally connecting mechanical device. This could be required ifmixing with bubbles, such as bubbles blown into the sample through thesensor, is not adequate or possible.

Addition F: Multiple volume metering arrangements can be successivelyincluded if the test must deal with different concentrations of thedifferent cells.

Addition G: A lid or coating tape may be used to cover the sample inletbefore use. This ensures a clean sampling area at the origination of thetest.

Addition H: A waste chamber may be applied at the outlet of thevolume-metering cell for waste or overflow of liquid.

Addition I: At any connection ports, e.g. the connection port to thepressure source, a small valve can be integrated to prevent liquid toleak out of the cartridge.

Addition J: A piston or membrane can be integrated into the cartridge toinclude a source of pressure for moving the liquid. The piston ormembrane could be moved by a mechanical force provided by theinstrument.

Addition K: An optical window can be integrated into the cartridge inorder to perform optical measurements such as spectrophotometricdetection of the haemoglobin content in a blood sample.

The methods described can be combined to give the best solution for thefinal application. The disposable sensor is particularly usable whereportable, cheap, simple or flexible equipment is needed, such as insmall laboratories, in measurements in the field or as a “point of care”(“near-patient”) diagnostic tool.

When using the Coulter principle the diluent for use in the apparatusaccording to the invention may contain inorganic salts rendering theliquid a high electrical conductivity. When sample is applied to theelectrolyte, the electrolyte to sample volumes should preferably behigher than 10. Sample preparation should preferably result in between1,000 to 10,000,000 cells per ml and more preferably between 10,000 and100,000 cells per ml. A mixing of the sample after adding electrolyte isrecommended. Cell diameters should preferably be within 1 to 60 percentof the orifice diameter and more preferably between 5 to 25 percent ofthe orifice diameter. Volume flow should preferably be from 10 μl to 10ml per minute and more preferably between 100 μl and 1 ml per minute.For the measurement a constant electrical current of approximately 1 to5 mA should preferably be applied. The source of electrical currentshould preferably have a signal to noise ratio (S/N) better than 1,000.The response from the electrodes can be filtered electronically by aband-pass filter.

According to yet another aspect of the invention a cartridge is providedcomprising a housing with a first mixing chamber and a first collectionchamber separated by a wall containing a first orifice for the passageof the cells between the first mixing chamber and the first collectionchamber, first cell characterization means for characterizing cellspassing through the first orifice, a bore in the outer surface of thehousing for entrance of the blood sample, communicating with a firstsampling member positioned in the housing for sampling the blood sampleand having a first cavity for receiving and holding the blood sample,the member being movably positioned in relation to the housing in such away that, in a first position, the first cavity is in communication withthe bore for entrance of the blood sample into the first cavity, and, ina second position, the first cavity is in communication with the firstmixing chamber for discharge of the blood sample into the first mixingchamber.

The cartridge may further comprise a second mixing chamber and a secondcollection chamber separated by a second wall containing a secondorifice for the passage of the cells between the second mixing chamberand the second collection chamber, second cell characterization meansfor characterizing cells passing through the second orifice.

In one embodiment of the invention, the first cavity is in communicationwith the first mixing chamber, when the first sampling member is in itsfirst position, for entrance of liquid from the first mixing chamberinto the first cavity, and, in a third position of the first samplingmember, the first cavity is in communication with the second mixingchamber for discharge of the liquid in the first cavity into the secondmixing chamber.

In another embodiment of the invention, the cartridge further comprisesa second sampling member positioned in the housing for sampling a smalland precise volume of liquid from the first mixing chamber and having asecond cavity for receiving and holding the sampled liquid, the memberbeing movably positioned in relation to the housing in such a way that,in a first position, the second cavity is in communication with thefirst mixing chamber for entrance of liquid from the first mixingchamber into the first cavity, and, in a second position, the secondcavity is in communication with the second mixing chamber for dischargeof the sampled liquid in the second cavity into the second mixingchamber.

The cartridge may further comprise a reagent chamber positioned adjacentto the first mixing chamber for holding a reagent to be entered into thefirst mixing chamber.

Preferably, the cartridge further comprises a breakable seal separatingthe reagent chamber from the first mixing chamber.

With this embodiment, different chemical treatment of different parts ofthe blood sample may be performed.

Also with this embodiment, further dilution of the blood sample may beperformed. The invention will be further described and illustrated withreference to the accompanying drawings in which:

FIG. 1 illustrates cell volume reduction after treatment by the lysingreagent according to the present invention,

FIG. 2 is a histogram of cell sizes in a blood sample after treatment bythe lysing reagent according to the present invention,

FIG. 3 shows a cross sectional side view through the components of adisposable unit 85, referred to as the cartridge,

FIG. 4 schematically illustrates the flow-through sensor concept

FIG. 5 illustrates an apparatus with a disposable cartridge, a dockingstation, and a reader,

FIG. 6 schematically illustrates the sampling procedure,

FIG. 7 schematically illustrates the cartridge and hydraulicconnections,

FIG. 8 schematically illustrates a second embodiment of the cartridge,

FIG. 9 schematically illustrates a third embodiment of the cartridge,and

FIG. 10 shows in perspective an apparatus according to the invention.

The elimination of the erythrocytes and the decrease in volume of theleukocytes and thrombocytes after exposure to the lysing reagent isshown in FIGS. 1 a (before lysing) and 1 b (after lysing). Further, ascan be seen in FIG. 2, the leukocytes decrease so as remains of them canbe found in the particle size interval of 4-7 μm. This should becompared to their original diameter of 8 to 20 μm. Also, according toFIG. 2, the largest remains of the thrombocytes have a diameter of 2.5μm, which should be compared to their original range in diameter of 2 to4 μm (ref. M. L. Stevens, Fundamentals of Clinical Haematology, W.B.Saunders Company, 1997).

EXAMPLE 1

Counting of thrombocytes, leukocytes, lymphocytes, monocytes andgranulocytes.

Use of a reagent mixture containing quaternary ammonium salts.

Material

3 fresh venous blood samples in (K₃)EDTA-collection tubes.

Coulter Particle Size and Particle Counter Z-2 equipped with a 50 μmorifice.

Lysing reagent mixture: 0.450 g 1,2,4-Triazole, 0.178 gDodecyltrimethylammonium chloride and 0.038 g Hexadecyltrimethylammoniumbromide were dissolved in 90 ml Diluide III-Diff (J. T. Baker). To thissolution 28.9 ml of de-ionised water was added. The final solution wasfiltered (0.22 μm).

Methods

The samples were first analysed for comparative purposes using anADVIA-120 (Bayer) system. Thereafter the blood samples were diluted 500times by adding 20 μl of them to 9.980 ml of lysing reagent mixture.Finally they were analysed on the Coulter Particle Size and ParticleCounter within 2 minutes after dilution.

Results

FIG. 2. shows a histogram for analysis of one of the blood samples. Inthis histogram the ranges used for correlation to the remains of thedifferent blood cells are shown. Identical ranges were used in theanalogous analysis of the remaining two blood samples.

TABLE 1 Blood Comparative sample Analyte Obtained result resultDeviation# 1 Thrombocytes  391 × 10e9  351 × 10e9 11 2 Thrombocytes  343× 10e9  359 × 10e9 −4.4 3 Thrombocytes  340 × 10e9  300 × 10e9 13 1Leukocytes 5.21 × 10e9 5.42 × 10e9 −4.0 2 Leukocytes 4.36 × 10e9 4.24 ×10e9 2.8 3 Leukocytes 5.33 × 10e9 5.35 × 10e9 −0.5 1 Lymphocytes 1.23 ×10e9 1.19 × 10e9 3.5 2 Lymphocytes 0.44 × 10e9 0.41 × 10e9 8.4 3Lymphocytes 1.03 × 10e9 1.14 × 10e9 −9.8 1 Monocytes 0.27 × 10e9 0.30 ×10e9 −10 2 Monocytes 0.16 × 10e9 0.14 × 10e9 15.7 3 Monocytes 0.36 ×10e9 0.40 × 10e9 −10 1 Granulocytes 4.09 × 10e9 3.94 × 10e9 3.7 2Granulocytes 3.92 × 10e9 3.69 × 10e9 6.2 3 Granulocytes 4.32 × 10e9 3.80× 10e9 14 #100 × (Obtained result − Comparative result)/Comparativeresult

EXAMPLE 2

Counting of thrombocytes, leukocytes, lymphocytes, monocytes andgranulocytes.

Use of a reagent mixture containing saponin.

Material

Two fresh venous blood samples in (K₃)EDTA-collection tubes.

Coulter Particle Size and Particle Counter Z-2 equipped with a 50 μmorifice.

Lysing reagent mixture: 2.063 g of Saponin (ACROS, prod. no. 41923100)was dissolved in 500 ml Diluide III-Diff (J. T. Baker). The finalsolution was filtered (0.22 μm).

Methods

The samples were first analysed for comparative purposes using anADVIA-120 (Bayer) system. Thereafter the blood samples were diluted 500times by adding 20 μl of them to 9.980 ml of lysing reagent mixture.Finally they were analysed on the Coulter Particle Size and ParticleCounter within 2 minutes after dilution.

Results

Identical ranges in the histogram (compare FIG. 2) were used in theanalogous analysis of the two blood samples.

TABLE 2 Blood Comparative sample Analyte Obtained result resultDeviation# 1 Thrombocytes  189 × 10e9  190 × 10e9 −0.5 2 Thrombocytes 284 × 10e9  283 × 10e9 0.4 1 Leukocytes 5.41 × 10e9 5.12 × 10e9 5.6 2Leukocytes 7.64 × 10e9 6.77 × 10e9 13 1 Lymphocytes 1.76 × 10e9 1.77 ×10e9 −0.8 2 Lymphocytes 1.64 × 10e9 1.64 × 10e9 0 1 Monocytes 0.25 ×10e9 0.24 × 10e9 4.2 2 Monocytes 0.22 × 10e9 0.26 × 10e9 −15 1Granulocytes 3.28 × 10e9 3.11 × 10e9 5.3 2 Granulocytes 5.47 × 10e9 4.88× 10e9 12 #100 × (Obtained result − Comparative result)/Comparativeresult

FIG. 3 schematically illustrates a disposable cartridge with a housing85 for blood analysis comprises a liquid storage chamber 1 containing aliquid diluent 11, a first sampling member 2 positioned in the housing85 for sampling a blood sample 8 and having a cavity 10 for receivingand holding the blood sample 8, the member 2 being movably positioned inrelation to the housing 85 in such a way that, in a first position, thecavity 10 is in communication with a bore 90 for entrance of the bloodsample 8 into the cavity 10 by capillary forces, and, in a secondposition, the cavity 10 is in communication with the liquid storagechamber 1 and a mixing chamber 3 for discharge of the blood sample 8diluted by the liquid diluent 11 into the mixing chamber 3. The mixingchamber 3 is separated by a wall containing an orifice 59 from and acollection chamber 5 for the passage of the blood sample 8 between themixing chamber 3 and the collection chamber 5. The wall containing theorifice 59 constitutes a part of a flow-through sensor 4.

A volume metering arrangement is connected to the collection chambercomprising a volume metering chamber 6 having the size of the volume tobe measured during the measurement with two connecting channels 12, 13of relatively diminutive internal volumes for registering liquid entryand exit by optical or electrical means, from the volume meteringchamber a channel 7 leads out to a connection port 67 where a pressurecan be applied.

FIG. 4 schematically illustrates, the flow-through sensor 4 has adividing wall 91 with a relatively thin passage 59 for the passage ofcells suspended in liquid. The passage serves as a sensing zone fordetection and measurement of the individual cells. The passage in thesensor may be formed as a count orifice for counting and sizing cells byan impedance method known as Coulter counting. Cells can be aspiratedthrough the orifice by pressure driven flow in either direction. When asaline or other electrolytic liquid solution is added to the chambers,the two chambers will be electrically isolated from each other exceptfor the route for current flow provided by the passage through theorifice.

FIG. 5 illustrates an apparatus with a disposable cartridge, a dockingstation, and a reader. The chambers on each side of the flow throughsensor have electrodes 34, 35 extending from an external terminal 61, 62through the base wall 63 of the disposable unit and into a configurationfacing the inside of its respective chamber. The cartridge is placed ina docking station 66 in a portable apparatus in order to carry out thetest. The docking station 66 has a cup shaped housing having a base 70and a circumambient sidewall 71. In the base 70 there are respectivespring loaded electrical connectors 64, 65 for contacting the terminals61, 62 of the cartridge automatically when the cartridge is received asa push fit into the docking station. There is also a conduit 68 passingthrough the base wall 70 aligned with the conduit 67 of the cartridge.Conduit 67 at its opening into the upper face of the wall 70 has a seal69, such as e.g. and O-ring for forming a gas tight connection with thelower face of the base wall 63 of the cartridge. A vacuum pump 72 isconnected by a line 73 to the lower end of the conduit 68. In amodification of the apparatus, the vacuum pump 72 can be reversed so asto apply positive gas pressure to the conduit 68. Schematicallyindicated at 74 are the further conventional components of a Coultercounter including all the electronic circuitry and display equipmentneeded for the operation of the apparatus.

FIG. 6 schematically illustrates the blood sampling operation. Theillustrated part of the cartridge 2 includes the liquid storage chamber83 for storing a diluent for diluting the sample and the first mixingchamber 77 for mixing the sample 84 and the diluent. This figureschematically illustrates a device for sampling a small and accuratevolume of liquid in accordance with the present invention. The device 10comprises a first member 86 with a first opening 87 for entrance of ablood sample into a bore 75 in the first member 86 and with a secondopening 76 for outputting the blood sample from the bore 75. The bore 75forms a capillary tunnel. The first opening 87 of the first member 86may be brought into contact with a liquid 8 (shown in FIG. 3), 84 to besampled so that the liquid 84 may flow through the first opening 87 intothe bore 75 and out of the second opening 76 by capillary attraction.The device 12 further comprises a sampling member 78 with a first cavity82 for receiving and holding the blood sample 84 and having a thirdopening 88 communicating with the first cavity 82. The first cavityforms a capillary tunnel with essentially the same diameter as the bore75. The sampling member 78 is a circular cylinder that is movablypositioned in relation to the first member 86. During sampling of theliquid, the sampling member 78 is positioned in the illustrated firstposition in relation to the first member 86 wherein the second opening76 is in communication with the third opening 88 so that sampled liquidmay flow through the second 76 and third opening 88 into the firstcavity 82 by capillary attraction. The third opening 88 may bedisconnected from the second opening 76 in a second position of thesampling member 78 in relation to the first member 86 so that the bloodsample 84 contained in the first cavity 82 is disconnected from the bore75.

The sampling member 78 is inserted into a third cavity 34 of the firstmember 86 for receiving and accommodating a part of the sampling member78. The sampling member 78 may be displaced between the first and secondposition along a longitudinal axis of the sampling member 78 that isalso substantially perpendicular to a longitudinal axis of the firstcavity 82. The sampling member 78 may also be rotatable about alongitudinal axis that is substantially perpendicular to a longitudinalaxis of the first cavity 82. In the first position, the first 75 andsecond 82 capillary tunnels extend along substantially the samelongitudinal centre axis.

In the illustrated embodiment the first member 86 is symmetrical and hasa fourth cavity 80 with openings 81, 79 opposite the bore 75, and thesampling member 78 has an opening 89 opposite the opening 88 so that, inthe first position, a capillary tunnel extends through the first 86 andthe second 78 member and communicates with the environment throughopenings 87, 79. Thus, air may escape from the capillary tunnel throughopening 79. Further, in the first position, a part of the liquidentering the first cavity 82 will leave the cavity 82 through opening 89thereby ensuring that the cavity 82 has been completely filled withliquid during liquid sampling eliminating the risk of sampling with areduced sample volume leading to low accuracy sampling.

FIG. 6 a illustrates the device 2 ready for receiving the liquid. InFIG. 6 b, a sample has entered into the capillary tunnel 82, and in FIG.6 c the sampling member 78 has been rotated into the second position forisolation of an accurate volume of the sample 84, and finally FIG. 6 dillustrates that the sample 84 has been washed out of the capillarytunnel 82 and into the first mixing chamber 77 by the diluent.

Example: The capillary tunnel forming the first cavity 82 may have alength of 8 mm and a diameter of 0.9 mm for containing a blood sample of5.089 μL.

Example: The capillary tunnel forming the first cavity 82 may have alength of 5 mm and a diameter of 0.5 mm for containing a blood sample of0.982 μL.

Example: The capillary tunnel forming the first cavity 82 may have alength of 3 mm and a diameter of 0.3 mm for containing a blood sample of0.212 μL.

FIG. 7 schematically illustrates an apparatus with a disposablecartridge holding a lysing reagent according to the present invention, adocking station, and a reader. In the following the operation of theapparatus for counting different types of white blood cells, namelymonocytes, lymphocytes, granulocytes, and thrombocytes and fordetermining haemoglobin content is explained.

The lysing reagent for selectively lysing red blood cells is added tothe diluent in the storage chamber 1. When the whole blood 8 is added tothe opening 58 of the first capillary section 15, the blood will bedragged in to the capillary and through the middle section 10 and lastsection 14 of the capillary. The last section of the capillary isconnected to a fill-chamber 43 for visually verification of the filling.The fill-chamber 43 is connected through a conduct 44 to open air.

The blood filled middle section of the capillary is part of a knob 2that can be moved to a second position, connecting the ends of thecapillary to two other conducts, a conduct 45 connected to the storagechamber 1 and a second conduct 40 connected to the first mixing chamber3 respectively. A third conduct 39 is leading from the first mixingchamber to a port opening 42 in the cartridge. The port opening isconnected through a counter port opening 37 in the apparatus, throughtubing 46 to a three-position valve 51 and directed through the twopositions of the valve to open air through second tubing 55 or through athird tubing 50 to the suction port of a membrane pump 47.

When the blood and diluent with reagent has been sucked into the firstmixing chamber, the blood can be mixed by blowing bubbles through theorifice of the sensor 4. The air pressure is applied through thecollection chamber 5, via a fourth conduct 12A, a small volume chamber6A, a fifth conduct 12B, a large volume chamber 6B and a sixth conduct 7directed to an opening port 41 in the cartridge. A counter port 36 inthe apparatus is connected through a fourth tubing 48 to a second threeposition valve 52, which has positions to direct to both vacuum througha fifth tubing 56 to the suction port of the membrane pump, or to theexhaust of the membrane pump, through a third two position valve 53 anda sixth tubing 49, the third valve having two positions for theconnection and for directing the pump exhaust to open air through aseventh tubing 54 respectively.

After mixing the diluted and lysed blood (red blood cells is removed) itis ready to be measured. The first mixing chamber is connected throughthe first valve to open air and the collection chamber is connectedthrough the second valve to the suction port of the pump. The exhaust ofthe membrane pump is connected through the third valve to open air. Asthe blood and diluent flows from the mixing chamber into the collectionchamber, an electrical connection between to counter electrodes 34 and35 placed in each chamber is established through the liquid. Cells arecounted and differentiated by size according to the Coulter principle.Through sizing of the cells, the cells can be distinguished andcategorised into different groups containing cells of a certain type.Thus, leucocytes can be differentiated into granulocytes, lymphocytesand monocytes. Furthermore, thrombocytes (platelets) can bedifferentiated from leucocytes as well. In order to determine theconcentration, the volume of the diluted blood, which has been counted,must be known. Since thrombocytes are approximately ten times asfrequent as leucocytes, it may be necessary to measure two differentvolumes. The thrombocytes are counted according to a small volumechamber 6A positioned between the collection chamber and the largervolume. By registering the liquid entry and exit at the inlet and outletof the small volume chamber respectively, the counting period will begiven. Registration of the liquid level is preferably done by an opticalreflectance measurement at the inlet 33 and at the outlet 32. The outletof the small volume chamber is also the inlet of the large volumechamber 6B. This chamber is used in connection with counting ofleucocytes. At the outlet of the large volume chamber, a third opticalreflectance measurement 31 is performed to register the exit of theliquid from this chamber.

After counting both leucocytes and thrombocytes the haemoglobin contentcan be measured by optical spectroscopy preferably through the middlesection of the large volume chamber 30.

The process of making a test by means of the present invention can becharacterized as:

-   -   1) Draw blood by using a lancet device    -   2) Pick up blood droplet by touching the blood to the cartridge        inlet    -   3) Mount cartridge in the instrument (instrument starts and runs        the test)    -   4) Read the result from the display    -   5) Remove and discard cartridge

FIG. 8 shows schematically another preferred embodiment of the cartridgeaccording to the invention. The illustrated cartridge has a first member104 for sampling blood. The member 104 is movably positioned in relationto the housing 100 between three positions, a first position for bloodsampling, a second position to connect the first storage chamber 103with the first mixing chamber 112, and a third position to connect thesecond storage chamber 105 with the second mixing chamber 110. The bloodis passed through the bore 122 into the first cavity of the member 104by capillary forces or by applying a vacuum at the end of the samplingchannel 111. A liquid blocking valve 116 is arranged after the firstsampling member to hinder passage of blood through the channel. Afterthe blood sampling, the sampling member is turned to the second positionand the sample is flushed into the first mixing chamber 112 by theliquid in the first storage chamber 103. In the first mixing chamber 112the sample is diluted 1:200 with the liquid in the first storage chamber103 and a fraction is blown back into the first cavity of the samplingmember 104, which is turned to the third position so that the dilutedsample is flushed into the second mixing chamber 110 by the liquid inthe second storage chamber 105. In the second mixing chamber 110 thesample is further diluted 1:200 to a total dilution of 1:40,000 with theliquid in the second storage chamber 105. A hemolysing reagent isinjected into the first mixing chamber 112 by a piston 115, which breaksa seal 118 between a reagent chamber 119 and the first mixing chamber112. After hemolysing the blood the 1:200 diluted sample is ready forcounting non-hemolysed white blood cells and for measuring haemoglobinby photometry. The white cells are counted by passing them through afirst orifice 113 and measuring the response by impedance cell countingover a first electrode pair 117, 120. A fixed volume is counted by afirst volume metering arrangement 107 connected to the first collectionchamber 114. A first overflow volume 106 is arranged after the firstvolume metering arrangement 107. The white blood cells can bedifferentiated by volume after adding the lysing reagent to the blood.The white cells can be grouped by volume into: Granulocytes, Monocytesand Lymphocytes. The three groups together yield the total white cellcount.

In the second mixing chamber 11, red cells and platelets are counted.The red cells and platelets are counted by passing them through a secondorifice 109 and measuring the response by impedance cell counting over asecond electrode pair 121, 125. A fixed volume is counted by a secondvolume metering arrangement 101 connected to the second collectionchamber 108. A second overflow volume 102 is placed after the secondvolume metering arrangement 101.

The embodiment may further comprise an additional optical detector forphotometric determination of the haemoglobin content. Referred to simplyas “total haemoglobin”, this test involves lysing the erythrocytes, thusproducing an evenly distributed solution of haemoglobin in the sample.The haemoglobin is chemically converted to the more stable and easilymeasured methemoglobintriazole-complex, which is a coloured compoundthat can be measured calorimetrically, its concentration beingcalculated from its amount of light absorption using Beer's Law. Themethod requires measurement of haemoglobin at approx. 540 nm where theabsorption is high with a turbidity correction measurement at 880 nmwhere the absorption is low.

FIG. 9 shows schematically another preferred embodiment of the cartridgeaccording to the invention. The illustrated cartridge has a first member104 for sampling blood. The member 104 is movably positioned in relationto the housing 100 between two positions, a first position for bloodsampling, and a second position to connect the first storage chamber 103with the first mixing chamber 112. A blood sample is passed through thebore 122 into the first cavity of the member 104 by capillary forces orby applying a vacuum at the end of the sampling channel 111. A liquidblocking valve 116 is arranged after the first sampling member to hinderpassage of blood through the channel. After the blood sampling, thesampling member is turned to the second position and the sample isflushed into the first mixing chamber 112 by the liquid in the firststorage chamber 103. In the first mixing chamber 112 the sample isdiluted 1:200 with the liquid in the first storage chamber 103.

The cartridge further comprises a second sampling member 124 positionedin the housing 100 for sampling a small and precise volume of liquidfrom the first mixing chamber 112 and having a second cavity 123 forreceiving and holding the sampled liquid, the member 124 being movablypositioned in relation to the housing 100 in such a way that, in a firstposition, the second cavity 123 is in communication with the firstmixing chamber 112 for entrance of a diluted sample from the firstmixing chamber 112 into the first cavity 123, and, in a second position,the second cavity 123 is in communication with the second mixing chamber110 so that the diluted sample is flushed into the second mixing chamber110 by the liquid in the second storage chamber 105. In the secondmixing chamber 110 the sample is further diluted 1:200 to a totaldilution of 1:40.000 with the liquid in the second storage chamber 105.A hemolysing reagent is injected into the first mixing chamber 112 by apiston which breaks a seal between a reagent chamber and the firstmixing chamber 112. The piston, seal and reagent chamber are not shownin FIG. 9. After hemolysing the blood the 1:200 diluted sample is readyfor counting non-hemolysed white blood cells and for measuringhaemoglobin by photometry. The white cells are counted by passing themthrough a first orifice 113 and measuring the response by impedance cellcounting over a first electrode pair 117, 120. A fixed volume is countedby a first volume metering arrangement 107 connected to the firstcollection chamber 114. A first overflow volume 106 is arranged afterthe first volume metering arrangement 107. The white blood cells can bedifferentiated by volume after adding the lysing reagent to the blood.The white cells can be grouped by volume into: Granulocytes, Monocytesand Lymphocytes. The three groups together yield the total white cellcount.

In the second mixing chamber 110, red blood cells and platelets arecounted. The red cells and platelets are counted by passing them througha second orifice 109 and measuring the response by impedance cellcounting over a second electrode pair 121, 125. A fixed volume iscounted by a second volume metering arrangement 101 connected to thesecond collection chamber 108. A second overflow volume 102 is placedafter the second volume metering arrangement 101.

The embodiment may further comprise an additional optical detector forphotometric determination of the haemoglobin content. Referred to simplyas “total haemoglobin”, this test involves lysing the erythrocytes, thusproducing an evenly distributed solution of haemoglobin in the sample.The haemoglobin is chemically converted to the more stable and easilymeasured methemoglobintriazole-complex, which is a coloured compoundthat can be measured calorimetrically, its concentration beingcalculated from its amount of light absorption using Beer's Law. Themethod requires measurement of haemoglobin at approx. 540 nm where theabsorption is high with a turbidity correction measurement at 880 nmwhere the absorption is low.

1. A cartridge for counting and discriminating a plurality of types ofblood cells in a blood sample in one counting operation, comprising ahousing with a first liquid storage chamber that has prestored therein alysing reagent that lyses erythrocytes while maintaining countingability of other blood cell types, wherein the lysing reagent contains asurfactant comprising a saponin, or wherein the lysing reagent comprisesa quaternary ammonium salt and further comprisesN-(1-acetamido)iminodiacetic acid to further assist the quaternaryammonium salt in minimizing debris stemming from hemolysed red bloodcells, a first mixing chamber and a first collection chamber separatedby a wall containing a first orifice for the passage of the cellsbetween the first mixing chamber and the first collection chamber, afirst cell characterizer that characterizes and counts the plurality oftypes of blood cells passing through the first orifice, the first cellcharacterizer including respective electrodes in the first mixingchamber and the first collection chamber, a bore in the outer surface ofthe housing for entrance of the blood sample, and a first samplingmember positioned in the housing for sampling the blood sample andhaving a first cavity for receiving and holding the blood sample, thefirst sampling member being movably positioned in relation to thehousing in such a way that in a first position, the first cavity is incommunication with the bore to receive and hold the blood sample, and ina second position, the first liquid storage chamber communicates throughthe first cavity with the first mixing chamber so that the blood sampleheld in the first cavity can be flushed with the lysing reagentprestored in the first liquid storage chamber into the first mixingchamber and mixed with the lysing reagent in the first mixing chamber.2. A cartridge according to claim 1, wherein the first liquid storagechamber further comprises compounds selected from the group consistingof N-(1-acetamido)iminodiacetic acid, procaine hydrochloride, and1,3-dimethylurea for stabilizing leukocytes during hemolysis of the redblood cells.
 3. A cartridge according to claim 1, wherein the firstliquid storage chamber further comprises compounds selected from thegroup consisting of N-(1-acetamido)iminodiacetic acid, sodium chloride,and sodium sulphate for adjusting pH-value and osmotic pressure of theliquid therein.
 4. A cartridge according to claim 1, wherein the firstliquid storage chamber further comprises compounds selected from thegroup consisting of 1,3-dimethylolurea and chlorhexidine diacetate forminimizing bacterial growth.
 5. A cartridge according to claim 1,wherein the first liquid storage chamber further comprises compoundsselected from the group consisting of potassium cyanide, tetrazole, andtriazole for converting haemoglobin species to an end product suitablefor spectrophotometric analysis.
 6. A cartridge according to claim 1,wherein the first liquid storage chamber contains inorganic saltsrendering the liquid therein as having high electrical conductivity. 7.A cartridge according to claim 1, wherein the lysing reagent comprises1,2,4-Triazole, dodecyltrimethylammonium chloride, andhexadecyltrimethylammonium bromide.
 8. A cartridge according to claim 1,wherein the other blood cell types are reduced in size, andconcentration thereof is determined by counting a representativefraction of respective cells.
 9. A cartridge according to claim 1,wherein the other blood cell types include lymphocytes, which areselectively reduced in size by the lysing reagent and can be counted ina cell counter.
 10. A cartridge according to claim 1, furthercomprising: a second mixing chamber and a second collection chamberseparated by a second wall containing a second orifice for the passageof the cells between the second mixing chamber and the second collectionchamber, a second cell characterizer that characterizes and counts theplurality of types of blood cells passing through the second orifice,wherein in the second position, the first cavity is in communicationwith the first mixing chamber so that liquid from the first mixingchamber can enter into the first cavity, and wherein in a thirdposition, the first cavity is in communication with the second mixingchamber the liquid in the first cavity can be discharged into the secondmixing chamber.
 11. A cartridge according to claim 1, furthercomprising: a second mixing chamber and a second collection chamberseparated by a second wall containing a second orifice for the passageof the cells between the second mixing chamber and the second collectionchamber, a second cell characterizer that characterizes and counts theplurality of types of blood cells passing through the second orifice,and a second sampling member positioned in the housing for sampling asmall and precise volume of liquid from the first mixing chamber andhaving a second cavity for receiving and holding the sampled liquid, thesecond sampling member being movably positioned in relation to thehousing in such a way that in a first position, the second cavity is incommunication with the first mixing chamber for entrance of liquid fromthe first mixing chamber into the second cavity, and in a secondposition, the second cavity is in communication with the second mixingchamber for discharge of the sampled liquid in the second cavity intothe second mixing chamber.
 12. A cartridge according to claim 1, furthercomprising a reagent chamber positioned adjacent to the first mixingchamber for holding a reagent to be entered into the first mixingchamber.
 13. A cartridge according to claim 12, further comprising abreakable seal separating the reagent chamber from the first mixingchamber.
 14. A cartridge according to claim 1, wherein a mixing memberis positioned in the first mixing chamber.
 15. A cartridge according toclaim 1, further comprising a sensor for characterization of the liquid.16. A cartridge according to claim 15, wherein the sensor forcharacterization of the liquid is adapted for spectrophotometriccharacterization of the liquid.
 17. A cartridge according to claim 1,wherein the first orifice has a diameter in the range from 30 μm to 100μm.
 18. A cartridge according to claim 17, wherein the diameter of thefirst orifice is in the range from 35 μm to 50 μm.
 19. A cartridgeaccording to claim 17, wherein the diameter of the first orifice is inthe range from 30 μm to 45 μm.
 20. A cartridge according to claim 19,wherein the diameter of the first orifice is in the range from 35 μm to40 μm.
 21. A cartridge according to claim 20, wherein the diameter ofthe first orifice is substantially equal to 40 μm.
 22. A cartridgeaccording to claim 1, wherein the other blood cell types includemonocytes, which are selectively reduced in size by the lysing reagentand can be counted in a cell counter.
 23. A cartridge according to claim1, wherein the other blood cell types include granulocytes, which areselectively reduced in size by the lysing reagent and can be counted ina cell counter.
 24. A cartridge for counting and discriminating aplurality of types of blood cells in a blood sample in one countingoperation, comprising a housing with a first liquid storage chamber thatholds a lysing reagent that lyses erythrocytes while maintainingcounting ability of other blood cell types, wherein the lysing reagentcontains a surfactant comprising a saponin, or wherein the lysingreagent comprises a quaternary ammonium salt and further comprisesN-(1-acetamido)iminodiacetic acid to further assist the quaternaryammonium salt in minimizing debris stemming from hemolysed red bloodcells, a first mixing chamber and a first collection chamber separatedby a wall containing a first orifice for the passage of the cellsbetween the first mixing chamber and the first collection chamber, afirst cell characterizer that characterizes and counts the plurality oftypes of blood cells passing through the first orifice, the first cellcharacterizer including respective electrodes in the first mixingchamber and the first collection chamber, a bore in the outer surface ofthe housing for entrance of the blood sample, and a first samplingmember positioned in the housing for sampling the blood sample andhaving a first cavity for receiving and holding the blood sample, thefirst sampling member being movably positioned in relation to thehousing in such a way that, in a first position, the first cavity is incommunication with the bore to receive and hold the blood sample, and ina second position, the first liquid storage chamber communicates throughthe first cavity with the first mixing chamber so that the blood samplecan be flushed with the lysing reagent from the first liquid storagechamber into the first mixing chamber, wherein the cartridge comprises apressure channel communicating with the first collection chamber, thepressure channel being adapted for coupling to a pressure source suchthat liquid flow through the first orifice can be controlled by pressureapplied by the pressure source.
 25. A cartridge for counting anddiscriminating a plurality of types of blood cells in a blood sample inone counting operation, comprising a housing with a first liquid storagechamber that holds a lysing reagent that lyses erythrocytes whilemaintaining counting ability of other blood cell types, wherein thelysing reagent contains a surfactant comprising a saponin, or whereinthe lysing reagent comprises a quaternary ammonium salt and furthercomprises N-(1-acetamido)iminodiacetic acid to further assist thequaternary ammonium salt in minimizing debris stemming from hemolysedred blood cells, a first mixing chamber and a first collection chamberseparated by a wall containing a first orifice for the passage of thecells between the first mixing chamber and the first collection chamber,a first cell characterizer that characterizes and counts the pluralityof types of blood cells passing through the first orifice, the firstcell characterizer including respective electrodes in the first mixingchamber and the first collection chamber, a bore in the outer surface ofthe housing for entrance of the blood sample, and a first samplingmember positioned in the housing for sampling the blood sample andhaving a first cavity for receiving and holding the blood sample, thefirst sampling member being movably positioned in relation to thehousing in such a way that in a first position, the first cavity is incommunication with the bore to receive and hold the blood sample, and ina second position, the first liquid storage chamber communicates throughthe first cavity with the first mixing chamber so that the blood samplecan be flushed with the lysing reagent from the first liquid storagechamber into the first mixing chamber, wherein the cartridge comprises apressure channel leading from the first mixing chamber and communicatingwith a first fluid connection port in the cartridge, the first fluidconnection port being adapted for coupling to fluid pressure through asecond fluid connection port of a docking station, such that liquid flowthrough the cartridge is controllable by the fluid pressure applied by apressure source within the docking station.